4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

The test chemical is not mutagenic at the highest tested concentration both in the presence and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0mMand S9-induced metabolic activation for 3 hours. The results showed no evidence of cytotoxicity after treatment with test chemical . Independently of tested test chemical concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that test chemical in the concentration of 0,0.5, 1.0, 2.5 or 5.0 mMdoes not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Remarks:

Experiment 2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Species / strain / cell type:

S. typhimurium, other: TA97, TA98 and TA100

Remarks:

Experiment 3

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

Experiment 4

Details on mammalian cell type (if applicable):

0.5 ml S9 mix

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

2.0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
3.0, 6.4, 32, 160 or 800 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-anthramine (+S9; all strains)

Remarks:

3

Details on test system and experimental conditions:

2.METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
3.METHOD OF APPLICATION: No data

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each dosage was tested on 5 parallel plates and all the tests were performed on two separate occasions.

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
4.METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: Experiment 1: 20 mins
Experiment 2: 60 mins
- Exposure duration:
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

2.A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.
3.The plates were observed for increase in the number of revertants/plate
4.Values for the mean revertants per plate and the mutagenic ratio (MR: mean revertants per plate/mean spontaneous revertants per plate) were determined. An MR of greater than two inferred a positive mutagenic response

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

3.Comparisons of the number of revertants per plate were done as t-tests after a square-root transformation of each number had been performed to give homogeneity of variance. The mean number of revertants for n plates at each dose level was calculated to be the squared value of the mean (y) of the square roots. The standard error of the mean was calculated as 2 y squared root s2/n where s2 is the pooled variance of all individual square root values

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test.
There was no reduction in colony count but reduction in bacterial background lawn was observed in treated concentrations 5.0 mg/plate (T8), 1.582 mg/plate (T7) and no reduction in colony count as well as in bacterial background lawn in treated concentrations (0.501 (T6) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0, 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
3.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The toxicity of the substances was tested in the tester strains at 10-7 dilutions. Based on the results obtained, the doses were selected for the main study

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No dataMenthol did not induce gene mutation in Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Study 2

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. T

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. B

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data.

The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

Conclusion

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Study 3

Ames assay was performed to determine the mutagenic nature of test chemical . The test chemical was studies for its mutagenic nature using Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 with and without S9 metabolic activation system. The test chemical was mixed with DMSO and used at dose level of 0, 6.4, 32, 160 or 800 µg/plate. Consurrent solvent and negative control chemicals were also included in the study. Test chemical at the highest dose of 800µg/plate showed a toxic effect on all bacterial strains. However, Test chemical did not induce gene mutation in Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Study 4

Ames assay was performed to determine the mutagenic nature of test chemical . The test chemical was studies for its mutagenic nature using Salmonella typhimuruim strains TA97, TA98 and TA100 with and without S9 metabolic activation system in experiment 1 with preincubation period of 20 mins and Salmonella typhimurium strain TA100 with S9 metabolic activation system in experiment 2 with preincubation period of 60 mins. Test chemical did not induce a MR response that was greater than 2 and no dose related response was noted. Based on the observations made, borneol did not induce gene mutation in Salmonella typhimuruim strains TA97, TA98 and TA100 in the presence and absence of S9 metabolic activation system. It also did not induce gene mutation in Salmonella typhimurium strain TA100 in the presence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration assay

Target gene:

No data

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Remarks:

Experiment 6

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 27-32 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

Experiment 7

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum
Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
- Properly maintained: yes by 4 day passages
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

lymphocytes: Human

Remarks:

Experiment 8

Details on mammalian cell type (if applicable):

- Type and identity of media: RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

6.0.00, 0.004, 0.008 and 0.016 mg/mL
7.At three different doses with 0.2 mg/mL being the maximum dose concentration
8.0, 0.1, 1 or 10 mM

Vehicle / solvent:

6.- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol
7.- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol
8.- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Untreated negative controls:

yes

Remarks:

Untreated cells served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

6.METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 22-25 hrs (without metabolic activation system)
- Expression time: 16-21 hrs (with and without metabolic activation system- Phase I and II)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 21-25 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations per cytotoxicity experiment were selected. Since there was cytotoxicity observed in the experiment I (0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL) and II (0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL), experiment III is conducted with further lower concentrations (0.004 (T7), 0.008 (T8) and 0.016 (T9) mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
7.METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 24 and 48 hrs
- Expression time (cells in growth medium): 24 and 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
8.- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Rationale for test conditions:

No data

Evaluation criteria:

6.A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
7.The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
8.The metaphase cells were observed for chromosomal aberrations

Statistics:

Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test

Species / strain:

lymphocytes: Human perpheral blood lymphocytes

Remarks:

Experiment 6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

Experiment 7

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Species / strain:

lymphocytes: Human

Remarks:

Experiment 8

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

6.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was slight precipitation observed at 1 mg/mL concentration.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Cytotoxicity was assessed at the concentrations of 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL at initial cytotoxicity experiment (Experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (Experiment II) was conducted with 0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (Experiment III) was conducted with further lower concentrations of 0.004 (T7), 0.008 (T8) and 0.016 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.04 (NC), 9.83 (VC), 8.40 (T7), 6.43 (T8), 5.19 (T9) and 8.53 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.98 (NC), 9.94 (VC), 8.24 (T7), 6.63 (T8), 5.10 (T9) and 8.54 (PC).

In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.016 was selected as highest concentration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.

Hence the concentrations selected for the main study are 0.004, 0.008 and 0.016 mg/mL. The main study was performed in two independent phases

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
7.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The maximum dose of each sample was selected
by a preliminary test in which the dose needed
for 50% cell-growth inhibition was estimated using a cell densitometer

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
8.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: 10mM-menthol was chosen as the highest concentration
because higher concentrations significantly
affected the growth of human lymphocytes in
phytohaemagglutinin-stimulated cultures

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No dataTEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: 10mM-menthol was chosen as the highest concentration
because higher concentrations significantly
affected the growth of human lymphocytes in
phytohaemagglutinin-stimulated cultures

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical is not mutagenic at the highest tested concentration both in the presence and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Study 6

This study was conducted to determine the chromosomal aberration induction potential of  the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 mg/mL mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Based on the observations made, the test chemical is at the highest tested concentration of 0.016 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

 Study 7

Chromosomal aberration study was performed to determine the mutagenic nature of test chemical . The cells were exposed to the test material at three different doses with 0.2 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-test chemical (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.Test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

Study 8

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of test chemical . The study was performed using lymphocytes isolated from the heparinized peripheral blood samples of 12 male and 12 female adult human non-smoking volunteers both with and withoutS9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0, 0.1, 1.0 or 10 mM. About 0.5-1.0 x 10⁶isolated lymphocytes were cultured in RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. Concurrent solvent and positive control chemicals were also included in the study. All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor and tested for statistical significance by the chi-square test. The combined percentage structural aberration rate for males and females in the solvent (DMSO) control was 1.76. Lymphocyte cultures treated with 10mM-menthol alone (10mM), had a rate of 2.11. This difference was statistically insignificant. The presence of S-9 in the culture did not influence the aberration frequency. Cultures grown in the presence of MMC showed a several-fold increase in chromosomal aberration frequency, thus validating the experimental conditions used. Based on these considerations,Test chemical did not induce chromosomal aberrations in human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Mammalian cell gene mutation study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Metabolic activation:

with

Metabolic activation system:

S9 liver microsomal fraction obtained from Aroclor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)

Test concentrations with justification for top dose:

0, 0.5, 1.0, 2.5 or 5.0mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: The test chemical was dissolved in ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation
DURATION
- Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.
DETERMINATION OF CYTOTOXICITYAfter being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
OTHER: No data available

Rationale for test conditions:

No data available

Evaluation criteria:

The number of mutant colonies were counted and compared to the negative and positive controls. The mutation frequency was calculated.

Statistics:

The Standard deviation (± SD) was calculated.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

No data available

Remarks on result:

other: No mutagenic potential

GENE MUTATION TEST

 

Table 1A.Effect of test chemical exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

 

 

	With S9
	with TG	without TG
Neg. control	0/4	808/4
Pos. control	0/4	819/4
0.5 mM	0/4
1.0 mM	0/4	752/4
2.5 mM	0/4	223/4
5.0 mM	0/4	426/4

 

 

Table 1B.Mutation frequencyin CHO cells after 3 hrs of exposure to test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

  

 

	With S9
Neg. control	N/A
Pos. control	N/A
0.5 mM
1.0 mM	N/A
2.5 mM	N/A
5.0 mM	N/A

 

 

 

Conclusions:

When CHO cells were exposed to the test chemical in the concentration of 0,0.5, 1.0, 2.5 or 5.0mM the results did not show any evidence of gene toxicity. Thus, the test chemical is considered to be negative for mutagenicity.

Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0mMand S9-induced metabolic activation for 3 hours. The results showed no evidence of cytotoxicity after treatment with test chemical . Independently of tested test chemical concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that test chemical in the concentration of 0,0.5, 1.0, 2.5 or 5.0 mMdoes not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames assay:

Study 2

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102. T

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. B

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data.

The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

Conclusion

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Study 3

Ames assay was performed to determine the mutagenic nature of test chemical . The test chemical was studies for its mutagenic nature using Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 with and without S9 metabolic activation system. The test chemical was mixed with DMSO and used at dose level of 0, 6.4, 32, 160 or 800 µg/plate. Consurrent solvent and negative control chemicals were also included in the study. Test chemical at the highest dose of 800µg/plate showed a toxic effect on all bacterial strains. However, Test chemical did not induce gene mutation in Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Study 4

Ames assay was performed to determine the mutagenic nature of test chemical . The test chemical was studies for its mutagenic nature using Salmonella typhimuruim strains TA97, TA98 and TA100 with and without S9 metabolic activation system in experiment 1 with preincubation period of 20 mins and Salmonella typhimurium strain TA100 with S9 metabolic activation system in experiment 2 with preincubation period of 60 mins. Test chemical did not induce a MR response that was greater than 2 and no dose related response was noted. Based on the observations made, borneol did not induce gene mutation in Salmonella typhimuruim strains TA97, TA98 and TA100 in the presence and absence of S9 metabolic activation system. It also did not induce gene mutation in Salmonella typhimurium strain TA100 in the presence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

Study 6

This study was conducted to determine the chromosomal aberration induction potential of  the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 mg/mL mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Based on the observations made, the test chemical is at the highest tested concentration of 0.016 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

 Study 7

Chromosomal aberration study was performed to determine the mutagenic nature of test chemical . The cells were exposed to the test material at three different doses with 0.2 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-test chemical (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.Test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

Study 8

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of test chemical . The study was performed using lymphocytes isolated from the heparinized peripheral blood samples of 12 male and 12 female adult human non-smoking volunteers both with and withoutS9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0, 0.1, 1.0 or 10 mM. About 0.5-1.0 x 10⁶isolated lymphocytes were cultured in RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. Concurrent solvent and positive control chemicals were also included in the study. All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor and tested for statistical significance by the chi-square test. The combined percentage structural aberration rate for males and females in the solvent (DMSO) control was 1.76. Lymphocyte cultures treated with 10mM-menthol alone (10mM), had a rate of 2.11. This difference was statistically insignificant. The presence of S-9 in the culture did not influence the aberration frequency. Cultures grown in the presence of MMC showed a several-fold increase in chromosomal aberration frequency, thus validating the experimental conditions used. Based on these considerations,Test chemical did not induce chromosomal aberrations in human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

Study 9

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0mMand S9-induced metabolic activation for 3 hours. The results showed no evidence of cytotoxicity after treatment with test chemical . Independently of tested test chemical concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that test chemical in the concentration of 0,0.5, 1.0, 2.5 or 5.0 mMdoes not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Study 10

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mMand without metabolic activation for 3 hours. The results showed evidence of cytotoxicity after treatment with test chemical in the concentration of 2.5 or 5.0 mM.Independently of tested test chemical concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that test chemical in the concentration of 0,0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.